A public antibody class recognizes an S2 epitope exposed on open conformations of SARS-CoV-2 spike

Delineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigate the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We show that ∼82% of SARS-CoV-2 S-reactive B cells harbor a naive phenotype, which represents an unusually high fraction of total human naive B cells (∼0.1%). Approximately 10% of these naive S-reactive B cells share an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.

Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
Ramos cell lines, HEK293T, and HEK293F were authenticated by the provider (ThermoFisher) and Ramos cells phenotype was validated by FACS.
All cell lines tested negative for mycoplasma.
No commonly misidentified cell lines were used in the study.
10 anonymous healthy donor samples from the Dutch national blood bank Sanquin. No information was disclosed on these individuals.
No specific and/or targeted recruitment is used in this study.
Patient consent was waived due to anonymized donation of blood for blood donation, blood products and research by the donors to the Dutch national blood bank Sanquin. No ethics board was involved as this is not required when donating blood and/or blood products at Sanquin.
Antigen-specific detection acquisition and sorting: General protocol: Frozen PBMC samples were first depleted for T cells using CD3 selection kit II (StemCell) according to the manufacturer's instruction. Enriched B cells or Ramos B cell lines were then stained in Eppendorf tubes with 50-100 !L of antigen probe cocktail for 30 min at 4°C, subsequently washed with FACS buffer (PBS supplemented with 1 mM EDTA and 2% fetal calf) and stained with the Live/DEAD dye together with MAbs coupled with fluorophores for FACS (Table S1)  Gating strategy together with CD27 and IgD, was centrifuged at 14,000g at 4°C for 10min and supernatant harvested. Barcoded antibody mix, anti-CD19-AF700, Live/DEAD dye, and labelled SARS-CoV-2 S (AF647 and BV421) were added to the cells and stained for 30min at 4°C. Cells were then washed twice and resuspended in FACS buffer before facs acquisition B cell activation experiments of Ramos B cells 4×106 cells/mL in RPMI10 were loaded with 1.5 !M of the calcium indicator Indo-1 (Invitrogen) for 30 min at 37°C, washed with Hank's Balance Salt Solution supplemented with 2 mM CaCl2, followed by another incubation of 30 min at 37°C. Antigen-induced Ca2+ influx of B cells was monitored on a LSR Fortessa by measuring the 379/450 nm emission ratio of Indo-1 fluorescence upon UV excitation. Following 30 s of baseline measurement, aliquots of 1×106 cells/mL were then stimulated for 210 s at RT with either 20 !g/mL, 10 !g/mL or 5 !g/mL of SARS-CoV-2 S or the equimolar amount presented on I53-50NPs. Ionomycin (Invitrogen) was added to a final concentration of 1 !g/!L to determine the maximum Indo-1fluorescence.
Binding assays to cell surface expressed CoV-S : HEK293T cells were transfected with full length S plasmid DNA (SARS-CoV-2 WT and variants, other epidemic and endemic CoVs, and Bat-CoVs) using Lipofectamine2000 (Invitrogen). Briefly, 0.5×106 cells/well were plated in a 6-well plate. After 24h, 4 !g DNA and 10 !L lipofectamine were mixed, incubated and added to each well. After 48 h, cells were harvested and pooled, and 5×104 cells were incubated in RPMI with 50 !g/mL of purified SARS-CoV-2 S-reactive MAbs for 1 h at RT. Cells were subsequently washed twice with PBS and stained for 30 min on ice and in the dark, in 50 !L of FACS buffer containing 1:1000 diluted PE-conjugated goat anti-human IgG (Biolegend). Cells were then washed twice with FACS buffer, fixed with 2% PFA and subsequently analyzed on BD LSRFortessa.
Antibody-dependent cellular trogocytosis: HEK293F cells (Invitrogen) at a density of 1×106cells/mL were transfected using SARS-CoV-2 S plasmid and PEImax (1 µg/µl) in a 3:1 ratio in OptiMEM. HEK293F cells were harvested 72 hours after transfection and their plasma membrane was stained with 10µM PKH26 (Sigma-Aldrich) dye in PBS, for 20 min (RT) with periodic mixing. Cells were washed twice with PBS and taken up in culture medium. THP-1 effector cells (ATCC) were stained intracellularly with 0.05µM carboxyfluorescein succinimidyl ester (CFSE, ThermoFisher) in PBS and incubate 20 min (RT) with periodic mixing. Cells were washed twice with PBS and taken up in culture medium.). PKH26 stained HEK293F cells were opsonized for 30 min at 37°C, with serial MAb dilutions. 2G12-IgG1, specific for HIV-1 gp120, was used as a negative control. After incubation, cells were washed and THP-1 cells were added to the HEK293F cells at a 2:1 effector:target ratio. Plates were centrifuged shortly to promote cell to cell contact and incubated 1 hour at 37°C. Afterwards, cells were washed and resuspended in PBS/2% FCS and aquired on FACS.
Antibody-dependant cellular phagocytosis Fluorescent neutravidin beads (Invitrogen) were incubated with biotinylated SARS-CoV-2 S-2P or RBD protein overnight at 4°C . Beads were subsequently centrifuged shortly and washed twice with PBS/2% BSA to remove unbound antigen and block the remaining hydrophobic sites on the microspheres. The coated beads were resuspended in PBS/2% BSA and 0.1 !L of the original suspension was placed in every well of a V-bottom 96 well plate and incubated (2 h at 37°C) with serial MAb dilutions. 2G12-IgG1, specific for HIV-1 gp120, was used as a negative control. After incubation, plates were washed and 5×104THP-1 effector cells (ATCC) were added to each well in a final volume of 100 !L of RPMI10. Subsequently, plates were centrifuged shortly to promote beads to cell contact before incubation (5 h at 37°C). After incubation, the cells were washed, resuspended in PBS/2% FCS and analyzed by flow cytometry.
Flow cytometry data were aquired using LSR Fortessa III and FACS ARIA II BD FACS DIVA software, Flowjo v10.7 software Purity of magnetically enriched B cells was confirmed by flow cytometry. Purity of sorted antigen-specific B cells for MAbs isolation and single cell RNA-seq could not be assessed due to the scarcity of the population, no post-sort acquisition was performed, all sorted samples were used for further analysis.
Combinatorial probe gating strategy: Conventionally, antigen-specific B cells are detected by the binding of two different fluorochrome-coded to the same protein.
In our study, we were able to detect 6 different antigen-specificities using 4 distinct fluorophores. By using a combinatorial probe staining strategy: SARS-CoV-2 S (AF647, BV421), H1N1 HA (BUV615, BV421), RSV F (AF647, BUV615), HCV E1E2 (AF647, BB515), HIV-1 ConM Env (BB515, BUV615) and Tetanus toxoid (BB515, BV421). For the analysis, the lymphocyte population was first gated based on the morphology (FSC-A/SSC-A) and doublets were removed. Next, dead cells and remaining CD4+ cells to avoid artefact binding of HIV-1 probes were first excluded within a dump channel and live antigen-specific B cells were studied in the CD19+population. To remove potential cross-reactive B cells to streptavidin, each probe combination was first gated on cells double negative for the two other channels (Fig. S1).
B cell sorting for MAb isolation The lymphocyte population was first gated based on the morphology (FSC-A/SSC-A) and doublets were removed. Dead cells and non-B cells were first excluded within a dump channel (CD3-/CD14-/CD16-). Live B cells (CD19+) that were double positive for the SARS-CoV-2 S protein (AF647 and BV421) were single cell-sorted. Using index sorting, sorted cells were evaluated for their expression of IgD and CD27 and characterized as naive B cells (IgD+ CD27-), Double negative (IgD-CD27-), Classical memory (IgD-CD27+) or Unswitched memory B cells (IgD+ CD27+) B cell sorting for 10xgenomics The lymphocyte population was first gated based on the morphology (FSC-A/SSC-A) and doublets were removed. Dead cells were first excluded. Live B cells positives for the SARS-CoV-2 S protein (AF647 and BV421) were bulk sorted